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Korean J Parasitol > Volume 30(3):1992 > Article

Original Article
Korean J Parasitol. 1992 Sep;30(3):191-199. English.
Published online Mar 20, 1994.  http://dx.doi.org/10.3347/kjp.1992.30.3.191
Copyright © 1992 by The Korean Society for Parasitology
Isolation and partial characterization of cysteine proteinase from sparganum
C Y Song,D H Choi,T S Kim and S H Lee*
Department of Biology, Chung-Ang University, Seoul 156-756, Korea.
*Department of Parasitology, Seoul National University College of Medicine, Seoul 110-460, Korea.
Abstract

A proteolytic enzyme was purified from the tissue extract of spargana (plerocercoids of Spirometra erinacei) by DEAE-Trisacryl M ion exchange chromatography and thiopropyl-sepharose affinity chromatography resulted in a 21-fold purification. The proteinase activity was assayed with a synthetic fluorescent substrate, carbobenzoxy-phenylalanyl-7-amino-4-trifluoromethyl-coumarin. SDS-polyacrylamide gel electrophoresis of the purified materials revealed a single 28,000 dalton band. Inhibitor profiles of the band indicated that it belonged to cysteine endopeptidases. It exhibited identical pH curves with optimum at pH 5.5, and 50% activity from pH 4.7 to 8. It could completely degrade collagen chains to three identical products. It also showed some activity on hemoglobin. Furthermore, the band on immunoblots was reactive to the sera of sparganosis patients. These results suggest that the proteolytic enzyme belongs to cysteine proteinase which plays a role in the tissue penetration. Also it may be used as the antigen for diagnosis of active sparganosis.

Figures


Fig. 1
Affinity chromatography with thiolpropyl-sepharose 4B. The supernatants were loaded onto DEAE-Trisacryl M chromatography, pooled, dialyzed with Sephadex G-25 and concentrated. The active peaks were loaded onto thiolpropyl-sepharose 4B, 0.1M Tris-HCl containing 0.3 NaCl, 1mM EDTA buffer (pH 7.5) and 2.0ml fractions (Fno) were assayed for activity on CBZ-phe-arg-AFC (○). Fractions were monitored for protein content (•) at 280 nm.


Fig. 2
10~20% gradient SDS-PAGE of purification steps of plerocercoid extracts. Lane A, active peak from thiolpropyl-sepharose 4B; Lane B, active peak from DEAE-Trisacryl M; Lane C, extracts; Lane D, molecular weight protein markers; phosphorylase B (94 kDa), albumin (67 kDa), ovalbumin (43kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20 kDa), α-lactalbumin (14kDa).


Fig. 3
Estimation of molecular weight of proteinases from Spirometra plerocercoid by AcA54 gel filtration(A) and SDS-PAGE(B).


Fig. 4
Effect of pH on cysteine proteinase activity from plerocercoid extracts.


Fig. 5
Degradation of collagen (upper) and hemoglobin (lower) by proteinases from plerocercoid extracts. Upper; Lane A, molecular weight protein marker; Lane B, collagen control; Lane C, 30 min; Lane D, 1hr. Lower; Lane A, molecular weight protein markers; Lane B, hemoglobin control; Lane C, 2hr; Lane D, 4hr.


Fig. 6
IgG reactivity to purified plerocercoid cysteine proteinase(▴) from patients with sparganosis. Lane A, molecular weight protein marker; Lane B, C, D, E and F, human sera of sparganosis; Lane G, normal human serum.

Tables


Table 1
Purification of cysteine proteinase from solube extracts of sparganum


Table 2
Effect of endopeptidase inhibitors on proteinase purified from sparganum extracts

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